To investigate the safety and tolerability of COVID-19 vaccines in people with epilepsy (PwE).In this multicentric observational cohort study, we recruited adult patients (age > 18 years old) with epilepsy who attended the Outpatient Epilepsy Clinic from 1st July to 30th October 2021. We administered to the patients a structured questionnaire and interview on demographic and epilepsy characteristics, current treatment, previous SARS-CoV-2 infection, vaccine characteristics, post-vaccine seizure relapse, other side effect, variation of sleep habits, caffeine, or alcohol intake. Seizure frequency worsening was defined as a ratio between mean monthly frequency post-vaccination and mean monthly frequency pre-vaccination superior to 1. Patients were categorized in two groups: patients with seizure frequency worsening (WORSE) and patients with seizure stability (STABLE).p = 0.003) and had a higher pre-vaccine seizure frequency (p = 0.009) compared with patients in the STABLE-group. Drug-resistant epilepsy was also associated with seizure worsening (p = 0.01). One-year pre-vaccination seizure frequency pattern demonstrated that patients in the WORSE-group had a higher frequency pattern (p < 0.001). Multivariate analysis of the vaccinated group showed that only the seizure frequency pattern  was significantly associated with seizure worsening.A total of 358 people participated with a mean age of 47.46 ± 19.04. Focal seizure (79.1%), generalized epilepsy (20.4%), and unknown types of epilepsy (0.5%) were detected among participants. In total, 31 (8.7%) people expressed that they were not willing to receive a COVID-19 vaccine; 302 patients (92.35%) did not experience an increase in the seizure frequency (STABLE-group) whereas 25 patients (7.65%) had a seizure worsening (WORSE-group). Post-vaccine seizures occurred mainly in the 7 days following the administration of the vaccine. Patients in the WORSE-group were treated with a mean higher number of anti-seizure medication (ASMs) (In our cohort of vaccinated PwE, only a little percentage had a transient short-term increase of seizure frequency. The present study demonstrates that COVID-19 vaccines have a good safety and tolerability profile in the short term in PwE. Coronavirus disease 2019 (COVID-19), caused by acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has become a global pandemic [Up to now, the approved COVID-19 vaccines have demonstrated in clinical trials to be effective and safe –5.According to literature, vaccines have been sporadically associated with neurologic complications including the occurrence of afebrile and febrile seizures, in particular in pediatric population , 7. SeizInternational League Against Epilepsy (ILAE) recommended that PwE should receive COVID-19 vaccine (https://www.ilae.org/patient-care/covid-19-and-epilepsy/covid-19-vaccines-and-people-with-epilepsy). Moreover, PwE, as a vulnerable category, were listed as a priority group in Italy as other European countries during COVID-19 vaccination campaign .Based on clinical trials, the However, real-world studies on PwE and safety profile of COVID-19 vaccination are underrepresented in the current literature.This study aims to examine the safety and tolerability of vaccinations against COVID-19 in PwE and their correlation with epilepsy features.The study is a multicentric observational retrospective cohort study conducted across the COVID-19 vaccination campaign. We consecutively recruited patients with a diagnosis of epilepsy according to the International League Against Epilepsy (ILAE) criteria who were admitted to the Outpatient Clinics from three Italian tertiary referral epilepsy centers  between 1st July and 30th October 2021.Four vaccines were approved in Italy at the time of the study: Pfizer/BNT162b2, Moderna/mRNA1273, AstraZeneca/AZD122/ChAdOx1 n-CoV-19, and the Janssen/Ad26.We included in the study patients > 18 years old, who gave informed consent to participate and had received two doses of vaccines, or who had received a single dose of vaccination because of a previous SARS-CoV-2 documented infection and in case of vaccination with Janssen vaccines/Ad26, and evaluated seizure frequency until 90 days from the first dose. Patients with mild cognitive impairment or mild-to-moderate intellectual disability were included in the study only if a caregiver was available for providing accurate information about the patient. Exclusion criteria were refusal to give informed consent and not fluent Italian speakers.We also collected data on PwE who were not vaccinated at the time of the visit and their reasons for not being vaccinated.Clinical data on epilepsy and COVID-19 vaccinations were obtained via a structured questionnaire and interview. The questionnaire comprised the following information: demographic , epilepsy characteristics , current treatment, including anti-seizure medication (ASMs), ASMs variation in the previous year, other concomitant treatments, previous SARS-CoV-2 infection, type of vaccine and date of vaccination, post-vaccine seizure relapse , other side-effect due to vaccination , variation of sleep habits, caffeine or alcohol intake.Regarding sleep habits, we calculated the sleep efficiency that is the percentage of time spent asleep while in bed, by dividing the amount of time spent asleep (in minutes) by the total amount of time in bed (in minutes). A normal sleep efficiency is 85% or higher .The questionnaire was administered by trained Neurologists, specialized in epilepsy.Furthermore, the Beck Depression Inventory (BDI)  was admiSeizures and epilepsy were classified according to the ILAE for both seizure type, epilepsy type, and etiology . Drug-reSeizure frequency worsening was defined as a ratio between mean monthly frequency post-vaccination and mean monthly frequency pre-vaccination superior than 1.Hence, patients were categorized in two groups: patients with seizure frequency worsening (WORSE) and patients with seizure stability .Non-vaccinated patients were asked to report their concerns about COVID-19 vaccination.n) and percentage.Statistical analysis was performed in multiple steps. Descriptive statistics were used to describe demographic and clinical features of the sample. Numerical variables were described using the following measures: mean and standard deviation. Categorical variables were presented as absolute number ; for categorical variables, we adopted the Pearson’s chi-square (χ2). The level of significance was set at p < 0.05.In order to compare numerical variables, we used a nonparametric test .The model for multivariate analysis was made choosing variables for the significance in the univariate comparison and for clinical relevance All statistics were performed using Statistical Package for Social Science (SPSS®) software version 22 .The study conforms to the ethical guidelines of the 1975 Declaration of Helsinki , as reflected in a priori approval by the institution’s human research committee at each participating study site. The study was approved by Ethic Committee of Fondazione Policlinico A. Gemelli IRCSS (ID 3076—protocol number: 0016460/21) as coordinator center.The primary outcome of this study is the evaluation of the variation of seizure frequency in PwE within 90 days after the administration of the COVID-19 vaccine.Secondary outcome measures included the assessment of the variables associated with seizure frequency worsening .In the period of study, a total of 403 patients were admitted to our outpatients’ clinics and screened for participation to the study. A total of 360 patients met the inclusion criteria and were asked to participate to the study Fig. . The finOut of 358 patients, 229 patients were in monotherapy: 90 patients 25.14%) were treated with levetiracetam, 51 patients (14.25%) with sodium valproate, 39 patients (10.89%) with carbamazepine, 25 patients (6.98%) with lamotrigine, 9 patients (2.51%) with oxcarbazepine, 8 patients (2.23%) with phenobarbital, 5 patients (1.4%) with lacosamide, and 2 patients with topiramate (0.56%). One hundred fourteen patients (31.84%) took a combination of ≥ 2 ASMs. Fifteen patients did not take ASMs (4.19%) , 43 patients received the Moderna/mRNA1273 (13.23%), 24 patients the AstraZeneca/AZD122/ChAdOx1 n-CoV-19 (7.38%), and one patient the Janssen/Ad26 (0.31%).Three hundred and two patients (92.35%) did not experience an increase in the seizure frequency (STABLE-group) whereas 25 patients (7.65%) had a seizure worsening (WORSE-group). Post-vaccine seizures occurred mainly in the 7 days following the administration of the vaccine (mean ± SD: 8.23 ± 8.19 days) and have never been associated with fever as well as with significant changes in sleep or alcohol intake habits. Patients in WORSE group did not experience any change in seizure type if compared to the pre-vaccine period. Only eight patients (2.45%) needed hospitalization for the recurrence of seizure after vaccination, but in the period examined, no cases of status epilepticus have been documented in our cohort. Breakthrough seizures  occurred in only two patients, in both cases after the second dose (Table p = 0.003) and had a higher pre-vaccine seizure frequency (p < 0.001) (Table p = 0.01)  Table . Drug-re1) Table .Table 2Up < 0.001)  Table .All the comparisons are summarized in Table p < 0.001) was significantly associated with seizure worsening .Sixty-six patients (20.18%) experienced at least one side effect after the first dose of vaccination, and 70 patients (21.41%) had at least one side effect after the second dose. The side effects were all mild and fleeting and included local skin reactions  and muscular pain and minor systemic side effects such as flu-like syndrome, fever, myalgia, arthralgia, fatigue, and headache. Thirty-one patients experienced fever after vaccination (7 after the first administration and 24 after the second dose). None of the aforementioned conditions required hospitalization.This multicentric observational study aimed to explore the safety and tolerability profile of COVID-19 vaccination in the short term in a large Italian cohort of PwE.Only a small percentage of PwE in our cohort (7.65%) experienced seizure worsening. These patients were affected mostly by a poor-controlled epilepsy, and/or a drug-resistant epilepsy, and were on ASMs polytherapy before vaccination.The duration of epilepsy as well as the age of the patients, on the other hand, did not affect the worsening of seizures. In our cohort, there were no cases of status epilepticus and the hospitalization for seizure worsening occurred only in eight patients.A recent Chinese multicentric study on 491 PwE demonstrated that fewer than 10% of the patients reported the occurrence of seizure after COVID-19 vaccination . In the The correlation between any vaccines and seizures, epilepsy, and/or epileptic encephalopathies has been comprehensively studied, in particular in pediatric population . DiphtheVaccines have not been related with afebrile seizures, and several studies and reviews concluded that immunizations do not trigger the onset of epilepsy . In our Regarding general side effects, several studies concluded that their occurrence is comparable between general population and PwE , 16.In our cohort, vaccine general side effects were mild and transient and occurred in approximately 20% of patients both after the first and second dose. These data were consistent with those reported in the general population . These sVaccine hesitancy in PwE still remains a global issue, being the fear of aggravating epilepsy the major concern .There are no known interactions between the ASMs and COVID-19 vaccines .The main strength of the study is the number of patients recruited and the multicenter design, which allow minimizing the effects of possible bias of selection. Another strength is the characteristic of the study cohort, which is highly representative of the population of patients with epilepsy.A limitation of the study is the self-reporting of seizures and side effect and the exclusion of patients with cognitive impairment and intellectual disability when a caregiver was not available.The present study demonstrates that COVID-19 vaccines have a good safety and tolerability profile in the short term in PwE, with a minor impact on seizure worsening.Given the extent of COVID-19 spread all over the world, it is essential to develop large-scale immunity in an effort to stop the pandemic. In our cohort of COVID-19-vaccinated epileptic patients, only a little percentage had a transient short-term increase in seizure frequency, with minimal impact on the healthcare system.Uncontrolled epilepsy was the most important predictive factor of seizure recurrence after COVID-19 vaccination. Vaccination per se does not represent a major risk of aggravating epilepsy with benefits outweighing risks.


The optimized formulation with minimized vs. and maximized absolute ZP was predicted successfully utilizing a numerical technique. EGA-EML-APA exhibited a significant cytotoxic effect with an IC50 value of 5.472 ± 0.21 µg/mL compared to the obtained value from the free drug 9.09 ± 0.34 µg/mL. Cell cycle profile showed that the optimized formulation arrested MCF-7 cells at G2/M and S phases. In addition, it showed a significant apoptotic activity against MCF-7 cells by upregulating the expression of p53, bax and casp3 and downregulating bcl2. Furthermore, NF-κB activity was abolished while the expression of TNfα was increased confirming the significant apoptotic effect of EGA-EML-APA. In conclusion, apamin-functionalized emulsomes have been successfully proposed as a potential anti-breast cancer formulation.Ellagic acid has recently attracted increasing attention regarding its role in the prevention and treatment of cancer. Surface functionalized nanocarriers have been recently studied for enhancing cancer cells’ penetration and achieving better tumor-targeted delivery of active ingredients. Therefore, the present work aimed at investigating the potential of APA-functionalized emulsomes (EGA-EML-APA) for enhancing cytototoxic activity of EGA against human breast cancer cells. Phospholipon Breast cancer is considered the most frequent form of malignancy in women and the leading cause of cancer mortality among women in the globe, claiming the lives of 685,000 women in 2020 ,2. BreasRecently, there has been increasing evidence that substances originating from natural sources possess dual advantages of having anticancer potential with reduced side effects . Many naEllagic acid (EGA), a member of the ellagitannin family, was discovered in various berries, including strawberries, cranberries, blackberries, raspberries and goji berries, as well as in grapes, pomegranates, nuts, green tea, and Eucalyptus globulus’s stem and bark ,9. It isVarious nanocarriers, including lipid-based, polymer-based and inorganic-based ones, are nowadays utilized in cancer therapy for achieving enhanced solubility, anticancer activity and targeted delivery ,20. BecaEmulsomes (EML) are novel lipid-based nanocarriers made up of solid lipid core surrounded by a phospholipid bilayer . They coVarious approaches have emerged for surface modification of nanocarriers used in cancer therapy with the aim of prolonging circulation time, enhancing cancer cells penetration and achieving better tumor-targeted delivery . AmongstTherefore, the present work aimed at exploring the potential of APA-functionalized emulsomes (EGA-EML-APA) as a peptide-mediated vesicular delivery approach for enhancing cytotoxic activity of EGA against human breast cancer cells. Central composite response surface design was implemented for optimization of the proposed emulsomal formulation with minimized vesicle size (VS) and maximized absolute zeta potential (ZP). The optimized formulation was tested for cytotoxic and proapoptotic activity against human breast cancer cells.Several nano-sized formulations such as liposomes, nanoemulsions, niosomes, proniosomes, nanoparticles and ethosomes have gained a lot of attention in the field of drug delivery. Nevertheless, emulsomes possess unique characteristics that impart potential advantages to this system compared to others. The most unique characteristic of emulsomes, compared to other vesicular systems including liposomes and niosomes, is the solid fat core enclosed by phospholipid. This allows for enhancing the solubility of poorly soluble drugs and entrapping high amounts of such drugs within the core. The enclosed drug exhibits prolonged drug release and consequently extended efficacy. Second, an emulsomes-based system showed adequate potential for targeting by virtue of their nano-size. Another reported advantage is surpassing the development of multi drug resistance, often associated with over expression of a cell membrane glycoprotein, which causes efflux of the drug from the cytoplasm and results in an ineffective drug concentration inside the cellular compartment . Accordi2 and the least PRESS, the vs. data fitted the quadratic model, while the ZP fitted the two-factor interaction (2FI) model. The adjusted R2 and the predicted R2 for each response exhibited appropriate coincidence with a difference of less than the permissible limit of 0.2 verifying the model validity. Moreover, the selected model for each response exhibited adequate precision value higher than the desirable value of 4 indicating appropriate signal-to-noise ratio. According to the previously computed parameters, the selected models could be adequately utilized for the exploration of the experimental design space.Fit statistical analysis results for the responses, namely, vs. and ZP are presented in Furthermore, verification of the goodness of fit of selected models was carried out via developing diagnostic plots illustrated in Invading cancerous tissues is considered a significant problem in the development of formulations for moieties with anticancer activity. This needs ongoing research to alter the features of drug delivery systems in order to improve tumor accessibility . Becausep = 0.0001). The lack of fit F-value of 6.32 (p = 0.1448) shows a non-significant lack of fit; thus, fitting of the observed vs. to the recommended model is ensured. The equation demonstrating the quadratic model in terms of coded factor was generated by the software is given as:In our study, the VS of the prepared EML ranged between 263.7 ± 7.9 and 601.8 ± 18.9 nm . On the p < 0.0001 for all terms). Furthermore, the interaction terms as well as the quadratic terms were also significant at the 95% level of significance. The perturbation graph, displayed in 1, X2 and X3 supports this finding. The most influential factor was PC: CH molar ratio as indicated by the highest coefficient of its linear term X1 in the developed equation, followed by PC: CH molar ratio, then the APA molar concentration. As per ANOVA results, all the linear terms belonging to the analyzed factors had a significant effect on the emulsomal vs. . The lack of fit F-value of 4.37 (p = 0.1997) reflects non-significant lack of fit; thus, the fitting of absolute ZP values to the proposed model is confirmed. The equation generated by the software demonstrates the suggested model for the ZP in terms of coded factor as given below:Based on Analysis of variance (ANOVA) for absolute ZP, the significance of the two-factor interaction model was confirmed, as depicted by the corresponding F-value of 44.69 (1 and X3 corresponding to PC: CH molar ratio and APA molar concentration exhibited a significant effect on absolute ZP (=p  0.0057 for X1 and p < 0.0001 for X3). Additionally, all the interaction terms were significant at p < 0.05. 1 and the negative sign of X3 coefficients support this observation. The observed interlink between increased negativity with the concomitant increase in PC: CH molar ratio could probably be a result of an increase in the proportion of a negatively charged phospholipid in the outer layers [The statistical analysis demonstrated that the linear terms Xr layers . On the Numerical optimization and the desirability techniques were implemented for predicting the optimized variables’ levels that, upon combination, could result in minimized vs. and maximized absolute ZP. The ramp graphs presented in The shape of the optimized EGA-EML-APA was visualized using TEM as illustrated in The optimized formulation showed high entrapment efficiency of 93.7 ± 4.1%. The high entrapment ability of the proposed formulation could be due to the solid lipid core enclosed by the lipid bilayer which aids in increasing the entrapping of poorly soluble active ingredients . It is w50 value for EGA-EML-APA in MCF-7 was 5.472 ± 0.21 µg/mL compared to 9.09 ± 0.34 µg/mL for EGA. Furthermore, there was no effective cytotoxic effect of blank EML-APA when compared with the EGA and EGA-EML-APA in MCF-7 cells. The observed cytotoxic activity of the EGA-EML-APA is in line with the previously reported cytotoxicity of EGA against breast cancer cells including MCF-7 cells [Breast cancer is the most commonly occurring cancer in women and the second most common cancer overall, specifically estrogen receptor positive (ER) breast cancer. Ellagic acid has been demonstrated to have antitumor and anti-apoptotic effects in several cancer cells . However-7 cells ,53. Howe-7 cells ,55,56.p < 0.05) (p < 0.05). These data suggest that EGA-EML-APA caused sequential cell cycle arrest at G2/M and S phases, followed by apoptosis, which is represented by the increase in apoptotic pre-G1 population. A previous report demonstrated a comparable ability of EGA to interfere with the cell cycle especially by increasing the population of cells at the pre-G1 phase [To determine the mechanism by which EGA-EML-APA exerts its cytotoxic influence against MCF-7 cells, the effect on cell cycle phases was investigated. While untreated MCF-7 cells exhibited quick growth properties, treatment of the cells with the EGA, and EGA-EML-APA resulted in altered cell cycle progression and increased cell fraction in the pre-G1 phase C–E. The  < 0.05) C,E. FurtG1 phase . Howeverp < 0.05). Necrotic cell death showed a similar pattern in which the most significant increase was associated with EGA-EML-APA , leading to a significant loss of MMP in comparison to raw EGA. Thus, an enhanced efficacy with EGA-EML-APA could be obviously shown and can be attributed to the lipophilic nature of the delivery system, which plays a major role in enhancing the delivery of EGA incorporated into this novel nanocarrier platform [Upon exposure to apoptotic stimuli, damage to the mitochondrial membrane might occur, resulting in the loss of MMP . Hence, platform .Bax, Bcl2 and casp3 was also examined to confirm the apoptotic activity of EGA-EML-APA. As depicted in Bcl2, casp3 expression compared to EGA treatment (p < 0.05), Bax levels and decreased Bcl-2 levels favor apoptosis [casp3, which is an apoptotic transcription factor [p53 by 8-fold compared to 5-fold increases associated with EGA   B. Theref® 90 G  was obtained as a gift sample from Lipoid GmbH .Ellagic acid (EGA), Cholesterol (CH), Tristearin (TS) and Apamin (APA) were purchased from Sigma-Aldrich Inc. . Phospholipon® 90 G: cholesterol molar ratio , Phospholipon® 90 G: tristearin weight ratio  and APA molar concentration  were considered as numerical independent variables. Vesicle size  and zeta potential  were investigated as responses. The coded levels of the independent variables, represented as –1, 0, +1 analogous to lower, middle and upper values respectively, along with the corresponding actual values for each variable are listed in 2), as well as the predicted residual error sum of squares (PRESS) statistics. The equations expressing the optimal model for the responses were then developed by the software. The significance of the variables on the measured responses was analyzed using analysis of variance (ANOVA) at p < 0.05. Perturbation and response surface plots were developed to display the influence of the explored factors as well as their interactions.Response surface methodology was implemented for the formulation and optimization of EGA-EML-APA. Specifically, three-level, three-factor, face-centred central composite experimental design (α = 1) was utilized in this study. PhospholiponA numerical optimization technique following a desirability approach was used for prediction of the optimized EGA-EML-APA formulation; the desirability function amalgamates both responses aiming at anticipating the optimized levels of investigated factors ,67. The ® 90 G, cholesterol and tristearin were solubilized in 15 mL of chloroform/methanol blend . Evaporation of the solvent blend was done under reduced pressure using Rotavapor  at 40 °C. By placing the formed film in a vacuum oven for 24 h, any organic solvent residual was eliminated. Hydration of the films was then achieved by mild agitation with 10 mL distilled water containing APA for 3 h at room temperature. The resultant dispersion was ultra-sonicated for two cycles, each of 45 s with a time gap of two minutes for vs. reduction [A previously reported method was utilized for the preparation of EGA-EML-APA . Accurateduction ,68. Mean vs. (z-average) as well as ZP of the proposed EGA-EML-APA were determined by light scattering and electrophoretic techniques, respectively, using Nano ZSP  at 25 ± 1 °C. Adequate dilution with distilled water was done prior to measurement. Data were expressed as the mean of five determinations. The optimized EGA-EML-APA were visualized using a JEOL GEM-1010  transmission electron microscope (TEM) at 80 kV. One drop of diluted formulation was placed on a carbon-coated grid, which was then left to dry at temperature of 25 ± 0.5 °C. Furthermore, the sample was negatively stained with 1% phosphotungstic acid and then dried for 20 min at room temperature before being visualized. Entrapment efficiency of the optimized formulation was performed by an indirect method. In addition, 1 mL of emulsomes dispersion was ultracentrifuged at 100,000 rpm for 1 h at 4 °C. The residue was washed twice with phosphate buffer (pH 6.8) and subjected to re-centrifugation for 1 h. The combined supernatant was diluted with phosphate buffer (pH 6.8), and analyzed using the reported HPLC method of assay with UV detection at 254 nm. The procedure was repeated thrice . t represents the amount of total ellagic acid; and EGAu represents the amount of unentrapped ellagic acid. The entrapment efficiency was calculated using the following equation:v/v in a humidified atmosphere with 5% CO2 incubator and at 37 °C. Human breast cancer cell line (MCF-7) was obtained from the American Type Culture Collection . The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, penicillin and streptomycin at a concentration of 1% 3 cells per well in 96-well plates and further treated for 24 h with each treatment in a concentration range of 0.4 to 100 µg/mL. EGA was dissolved in DMSO . The culture media were then aspirated and replaced with MTT solution at a final concentration of 2 mg/mL. The cells were subsequently incubated for 3 h at 37 °C. Thereafter, removal of the MTT solution was performed, then 100 μL of DMSO was added in order to dissolve the formazan crystals. Measurement of the absorbance was then carried out at a 570 nm wavelength using an Absorbance-based Spark® multimode microplate reader . The results were represented as a percentage of cell viability relative to the control. Fitting of the dose response curves followed by calculating IC50 values was undertaken using GraphPad prism software .As directed by the manufacturer, the cytotoxic effect of EGA, blank EML-APA and the optimized EGA-EML-APA on MCF-7 cells was evaluated using the MTT viability test . The cells were grown overnight at a density of 5 × 105 cells/well were seeded in a 6-well plate prior to their incubation for 48 h with each treatment. Subsequently, the cells were rinsed, trypsinized and then centrifuged for 15 min at 1000× g. The cells were then fixed using 70% ethanol and incubated at 4 °C for 2 h. The fixed cells were then centrifuged at 1000× g for 15 min prior to staining with PI (10 g/mL) and RNase treatment. A flow cytometer  was used to assess cell DNA content, with a minimum of 20,000 events obtained for each treatment. Analysis of the data was done using the CellQuest Software .Flow cytometry analysis was applied to analyze the effects of EGA, blank EML-APA, and the optimized EGA-EML-APA on MCF-7 cell cycle phases using propidium iodide (PI) Flow Cytometry Kit . In addition, 3 × 106 cells per well and incubated for 48 h with each treatment. After that, the cells were trypsinized and subjected to centrifugation at 10,000× g for 5 min. The supernatant was then discarded; and the cells were rinsed in PBS and stained in dark conditions for 30 min using annexin V-FITC/PI dyes, as directed by the manufacturer. FACScalibur  was used to evaluate stained cells, and a minimum of 20,000 events were obtained for each treatment.The Annexin V-FITC Apoptosis Detection Kit  was used to investigate the effects of EGA, blank EML-APA and the optimized EGA-EML-APA on the apoptosis profile of MCF-7 cells. Briefly, in a 6-well plate, cells were plated at a density of 1 × 105 cells per well in 96-well plates. Cells were stained using 20 M tetramethylrhodamine, methyl ester (TMRM) following 48 h of incubation with EGA, blank EML-APA, or optimized EGA-EML-APA and then incubated for a period of 30 min at 37 °C. TMRM is a fluorescent, cell-permeant dye that creates a strong bright signal upon its accumulation in active healthy mitochondria of cells. TMRM buildup ends and the signal disappearing indicates that the mitochondrial membrane potential is lost during apoptosis. The cells were subsequently washed in PBS, loaded with live-cell imaging buffer and then analysed using flow cytometry .The determination of MMP was carried out as per the manufacturer’s directions using the MitoProbeTM TMRM Assay Kit for Flow Cytometry . MCF-7 cells culture was performed at a density of 1 × 10Qiagen’s RNeasy mini kit  was used to extract RNA from MCF-7 cells as per the manufacturer’s instructions. A Nanodrop spectrophotometer  was used to confirm the concentration and purity of RNA. A ratio of A260 nm/A230 nm, not smaller than 1.8, and a A260 nm/A280 nm ratio, not smaller than 1.9, were detected in all RNA samples.® Green kit . Relative expression patterns of p53, bcl2, bax casp3, tnfα and nf-κb were done utilizing 10 ng of RNA template in a 50-mL reaction mixture of iScript one-step RT-PCR kit with SYBR® Green mix using a 7500 Fast real-time PCR system . RNA was normalized between samples and reverse transcribed to complementary DNA (cDNA) as per the manufacturer’s instructions using the iScriptTM One-Step RT-PCR Kit With SYBRFace-centred central composite design has been successfully employed for optimizing EGA-EML-APA with minimized vs. and maximized absolute ZP. The use of APA in formulating the emulsomes was proposed to provide additional advantage of enhancing uptake of EGA by cancerous cells, in addition to the cytotoxic activity of bee venom components including apamin. The measured responses of the optimized formulation were 267.6 nm for vs. and −32.1 mV for ZP. The measured responses coincide well with the predicted ones, confirming the validity of the numerical optimization adopted in this study. The present work confirmed the increased percentage of cytotoxic activity and apoptosis of EGA against the human breast cancer cell line (MCF-7) after the formulation EML-APA. This was confirmed by observations in cell viability (MTT assay) as well as the changes in the expressions of apoptotic markers and inhibiting NF-κB activity. Arresting G2/M and S cell cycle phases by EGA-EML-APA is a superior finding with respect to the mechanism by which the new formulation is inhibiting MCF-7 cell proliferation. These data demonstrate that the proposed formulation could be a successful delivery platform to enhance the cytotoxicity of EGA against MCF-7 cells. Thus, our study established the in vitro efficacy of optimized EGA-EML-APA as a potential anti-breast cancer formulation. Further in vivo investigation of such formulation following administration via non-invasive para-enteral routes compared to intravenous and oral ones will be considered in future work.


Compared with the low plasma DKK-1 without significant coronary obstruction group, the high DKK-1 with obstructive CAD group had a hazard ratio of 10.640  for MACEs, as determined by multivariable Cox proportional hazard regression analysis. In conclusion, we observed a synergistic effect between high plasma DKK-1 and obstructive CAD on the prediction of MACEs in patients with angina.The canonical β-catenin-dependent wingless (Wnt) pathway is associated with endothelial function. We examined the effect of plasma dickkopf-1 (DKK-1), an inhibitor of the Wnt pathway, on the prediction of major adverse cardiac events (MACEs). We enrolled patients who had undergone selective coronary angiography for angina. DKK-1 levels were determined using plasma collected at the outpatient visit after fasting. MACEs served as the primary endpoint. All 470 enrolled patients were divided into four groups according to their median plasma DKK-1 levels and the presence of obstructive coronary artery disease (CAD). Forty-eight patients reached the primary endpoint during a median follow-up time of 4.8 years. Kaplan–Meier survival analysis indicated that the group with high DKK-1 and obstructive CAD had a significantly higher mortality rate than the other three groups (log-rank test  The global number of patients with ischemic heart disease (IHD) is increasing, and IHD remains a major health burden worldwide ,2. AccorDickkopf-1 (DKK-1) is a secreted glycoprotein that acts as an inhibitor of the canonical β-catenin-dependent wingless (Wnt) pathway . Wnt proAdmission serum levels of DKK-1 in patients with acute coronary syndrome have been reported to be associated with major adverse cardiac events (MACEs), a composite of cardiovascular death, myocardial infarction (MI), or stroke . DKK-1 iThis observational cohort study was conducted at Taichung Veterans General Hospital between January 2010 and March 2018. The study complied with the guidelines of the Declaration of Helsinki and was approved by the Institutional Review Board of Taichung Veterans General Hospital. Written informed consent was obtained from every participant before the start of any procedure. The inclusion criteria for patient enrollment were (a) a history of angina, (b) a diagnosis of IHD based on at least a noninvasive examination, and (c) a selective coronary angiography performed in our hospital. The exclusion criteria were as follows: (a) age < 20 years, (b) a history of known diabetes mellitus (DM), (c) a history of known cancer, (d) current medication for any autoimmune disease or any psychiatric disease, (e) current acute infection, (f) symptomatic congestive heart failure ≥ Class III based on the criteria of the New York Heart Association , and (g)p = 0.351).After patients were discharged in a stable condition, the eligible patients were scheduled for an outpatient follow-up appointment. We enrolled 470 patients between January 2010 and December 2016. Following anthropometric measurements in the outpatient interview, fasting blood samples were collected. We recorded the coronary angiography and medications by reviewing electronic medical records. After the baseline assessment, all of the participants were followed up through March 2018. Death due to any cause or the first episode of nonfatal MI or nonfatal stroke was recorded as the primary endpoint of MACEs based on electronic medical records from our hospital. For patients without any endpoint recorded before February 2018, we arranged a telephone call interview in March 2018, and information on the primary endpoint was collected from the patients themselves or from their immediate family members. Among all of the enrolled participants, 431 (91.7%) completed follow-up, and the data accuracy was confirmed using death registration information obtained from the Ministry of Health and Welfare, Executive Yuan, Taiwan. In the 39 (8.3%) participants who were lost to follow-up, we confirmed death using death registration information. There was no significant difference in the mortality rate between the participants who were followed up and those who were lost to follow-up , and DKK-1 levels. Human DKK-1 was measured using a commercial immunoassay kit . The precision of the DKK-1 measurements was indicated by an intra-assay coefficient of variation (CV) of 2.6% and an inter-assay CV of 8.1%. The minimum detectable level of the DKK-1 measurement was 0.419 pg/mL. Platelet counts were determined using an automated cell counter . Human NT-proBNP was measured using a commercial immunoassay kit . Serum samples were prepared to measure creatinine levels, C-reactive protein (CRP), and the lipid profile. CRP was determined using an ELISA kit . A morning urine sample was collected for the measurement of albumin and creatinine to calculate the urine albumin to creatinine ratio (UACR).−1.154 × (age [years])−0.203  [2 [The homeostasis model assessment of insulin resistance (HOMA-IR) index was calculated using the following formula: fasting insulin (µIU/mL) × fasting glucose (mmol/L)/22.5 for the quantitative evaluation of insulin resistance . Accordimale) [2 . CAD wasContinuous variables are presented as the mean ± standard deviation, and we used the Kolmogorov–Smirnov test to examine the normality of the data. We used the t-test or the Mann–Whitney U test between two groups and analysis of variance (ANOVA) or the Kruskal–Wallis test among more than two groups to ascertain the significance of the difference in the continuous variables. Categorical variables are presented as numbers (with percentages). We used chi-squared tests to ascertain the significance of the difference in the categorical variables. The risk of MACEs was examined by Kaplan–Meier survival analysis among the four groups, and statistically significant differences were tested by the log-rank test. The improvement in primary-endpoint prediction by adding the variable of DKK-1 was assessed by examining the increases in Harrell’s concordance index (C index) . We usedp value < 0.05 was considered to be statistically significant. Statistical analysis was conducted using SPSS v22.0  and R software v3.4.Multivariable Cox proportional hazards regressions were performed to evaluate the risk of MACEs according to the groups categorized by plasma DKK-1 level and CAD status. A two-sided p = 0.622; p = 0.146). Among the 470 patients enrolled in the present study, there were 187 patients without CAD and 283 patients with CAD. There was no significant difference in the plasma DKK-1 levels between patients without CAD and those with CAD , the proportion of current smokers (p = 0.044 among the four groups), and serum CRP (p = 0.003 among the four groups) from the low DKK-1 without CAD group to the high DKK-1 with CAD group. There were higher proportions of males (p < 0.001 among the four groups), hypertension (p = 0.033 among the four groups), and the use of statins (p < 0.001 among the four groups) in the patients with CAD than those without CAD. The eGFR (p < 0.001) and lipid profile, including total cholesterol (p < 0.001), high-density lipoprotein (HDL) cholesterol (p < 0.001), and triglycerides (p = 0.042), were significantly different among these four groups.The baseline characteristics of these four groups are shown in p = 0.001).During the median follow-up time of 4.8 years, a total of 48 MACEs occurred. All the patients were divided into four groups : 94 patip = 0.027) and a significant NRI of 0.215  compared with the FRS and CAD model.We calculated the C index to compare the performance of MACE prediction among the different models. The C indices of the FRS-alone model, the FRS and CAD model, and the model adding DKK-1 to FRS and CAD were 0.64, 0.66, and 0.69, respectively . The modp = 0.025) after adjusting for age, sex, smoking status, hypertension, HOMA-IR index, total cholesterol, HDL cholesterol, triglycerides, eGFR, platelet count, CRP, NT-proBNP, UACR, and the use of statins.We conducted multivariable Cox regression, and the results are presented in The main finding of the present study is that a combination of plasma DKK-1 levels and obstructive CAD acted as a better predictor of MACEs than plasma DKK-1 alone or obstructive CAD alone for a median of 4.8 years of follow-up in these patients. In accordance with our findings, the DKK-1 levels in serum have been reported to be a significant predictor of MACEs in patients after acute coronary syndrome. In a population-based cohort study, Klingenschmid et al.  reportedThere have been discordant results regarding the association between plasma DKK-1 and CAD in previous studies: a higher plasma DKK-1 level was reported in patients with stroke than in healthy controls ; howeverThe Wnt signaling pathway plays an important role in cardiovascular development and regeneration . The abeThe endothelium is an important source of DKK-1 secretion. According to in vitro studies using human umbilical vein endothelial cells (HUVECs), the induced overexpression of DKK-1 in the endothelium could subsequently trigger monocyte adhesion to the endothelium, apoptosis of the endothelium, and a rupture risk for vulnerable plaques ,27. MoreAlthough angina is a clinical symptom of IHD ,31, signThere are several limitations in the present study. First, we did not directly assess the underlying mechanism involved in DKK-1 and MACEs in patients with angina. Second, we did not examine microvascular dysfunction using invasive stress tests in the enrolled patients who did not present with significant coronary obstruction . Third, Plasma DKK-1 levels in patients with angina and significant coronary obstruction were not significantly different from those in patients with angina but no significant coronary obstruction. However, higher plasma DKK-1 levels and obstructive CAD had a synergistic effect on predictions of a higher risk of MACEs in patients with angina during a median follow-up of 4.8 years.


TOPK/PBK (T-LAK Cell-Originated Protein Kinase) is a serine/threonine kinase that is highly expressed in a variety of human tumors and is associated with poor prognosis in many types of human malignancies. Its activation mechanism is not yet fully understood. A bidirectional signal transduced between TOPK and ERK2  has been reported, with ERK2 able to phosphorylate TOPK at the Thr9 residue. However, mutated TOPK at Thr9 cannot repress cellular transformation. In the present study, Ser32 was revealed to be a novel phosphorylated site on TOPK that could be activated by ERK2. Phospho-TOPK (S32) was found to be involved in the resistance of renal cell carcinoma (RCC) to sorafenib. Herein, combined a TOPK inhibitor with sorafenib could promoted the apoptosis of sorafenib-resistant RCC. High expression of HGF/c-met contributes to activation of p-TOPK (S32) during the development of sorafenib resistance in RCC. The current research presents a possible mechanism of sorafenib resistance in RCC and identifies a potential diagnostic marker for predicting sorafenib resistance in RCC, providing a valuable supplement for the clinically targeted treatment of advanced RCC. RCC account for 2.7% of adult malignancies , 2. ApprTOPK is a member of the MEK family and is highly expressed in many tumor types –15. BidiIn this study, we identified Ser32 as a novel site on TOPK that can be phosphorylated by ERK2. We also verified that TOPK is activated at Ser32 during the development of sorafenib resistance and revealed the possible molecular mechanism in advanced RCC. We also explored sorafenib combined with a TOPK inhibitor as a treatment strategy for sorafenib-resistant mRCC. These investigations will be beneficial for the increasing number of sorafenib-resistant patients when the drug is used as a treatment for mRCC.https://portal.gdc.cancer.gov/) were utilized to analyze the correlation between TOPK expression and clinical stage, as well as the pathological grade of renal carcinoma and the relationship between expression and overall survival (OS) of patients. All analyses were performed using the R project. The scatter plot of TOPK expression in RCC compared to that in normal kidneys  and can be used as an independent prognostic factor in kidney cancer  and stage IV (p < 0.001) RCC    database  Fig. . The exp23) Fig. . Based o32P] ATP in a kinase assay in vitro. Results indicated that peptide S3 (include Ser32) was phosphorylated by active ERK2 more strongly than any other residue  found on serine 13 and serine 32, respectively, among the fifteen residues Fig. . All fifdue Fig. .Fig. 2ERTo verify the above results, a TOPK plasmid was constructed with TOPK 1-31 deleted, where the TOPK protein was expressed and purified from bacteria, and a kinase assay in vitro was performed. Results revealed that TOPK was still activated by ERK2 even in the absence of the T9 residue Fig. . InactivThe experiments of bonding between TOPK and ERK1/2 was not repeated because such results had been reported. Different quantities of ERK2 plasmid  were transfected into HEK293 cells, and then cells were stimulated with EGF (40 ng/ml). Endogenous phosphorylation of TOPK S32) was then analyzed by western blotting. Results indicated that increasing the amount of transfected ERK2 plasmid resulted in an increase in endogenous p-TOPK (S32)  on carcinogenesis, wild type TOPK (TOPK-WT) was mutated to TOPK-S32A. The wild type or mutant TOPK were overexpressed in the JB6 Cl41 (JB6 for short) and Caki-1 cell lines respectively Fig.  . JB6 isThe resistance of sorafenib in RCC is still an unresolved problem. Here, we further explore whether p-TOPK (S32) is involved in this process. First, the responsiveness of the RCC cell lines to sorafenib was determined by cell viability (%) assays, and results indicated that 786-O and 769-P cells were sensitive to sorafenib, while ACHN and Caki-1 cells were resistant to sorafenib Fig. . Next, t786-O, 786-O-SR and ACHN cells were cultured and incubated with different concentrations of sorafenib, and expression of p-TOPK (S32) and p-c-Raf were determined by western blot. We found that the expression of p-TOPK (S32) in the 786-O cell line was inhibited by sorafenib in a concentration-dependent manner Fig. . The expTOPK was stably knocked down in 786-O-SR and ACHN cells with lentiviral infection Fig. . The respcDNA3, pcDNA3-TOPK and pcDNA3-TOPK-S32A were overexpressed in 786-O cell line respectively Fig. . After tBased on the above results, we investigated whether inhibition of TOPK would suppress sorafenib-resistant RCC cells and restore RCC cell sensitivity to sorafenib. OTS964 is a TOPK inhibitor, with better pharmacokinetic profile for in vivo work than others –22. 786-A diagram of the in vivo experimental timeline is shown in Fig. HGF/c-Met was reported to be a driver and biomarker of VEGFR inhibitor resistance in non-small cell lung cancer  and to cRas/Raf/MEK/ERK pathway is closely related to tumorigenesis and TKI resistance in different types of cancer . TOPK haet al. reported that the HGF/c-MET pathway mediates VEGFR inhibitor resistance and vascular remodeling in NSCLC  and an internal control GAPDH mRNA .supplementary data fileTable S1Newly added co-author confirmation letteraj-checklist


PNI-U-DPy exhibits characteristics similar to PNIPAM but could form micelles even when the environment temperature is lower than its LCST. The U-DPy moieties could utilize their strong complementary hydrogen bonding–interaction to create a physically crosslinked network within PNIPAM micelles, thus adjusting its LCST to a value near the physiological temperature. Morphological studies suggested that C60 could be effectively loaded into PNI-U-DPy micelles with a high loading capacity (29.12%), and the resulting complex PNI-C60 is stable and remains temperature responsive. A series of measurements under variable temperatures was carried out and showed that a controlled release process proceeded. Furthermore, PNI-C60 exhibits hydroxyl radicals scavenging abilities at a low dosage and could even be adjusted by temperature. It can be admitted that the micelle system can be a valuable alternative for radical scavengers and may be delivered to the desired position with good dispersibility and thermo-responsivity. It is beneficial to the search progress of scientists for drug delivery systems for chemotherapeutic treatments and biomedical applications.Living creatures involve several defense mechanisms, such as protecting enzymes to protect organs and cells from the invasion of free radicals. Developing antioxidant molecules and delivery systems to working with enzymes is vital. In this study, a supramolecular polymer PNI-U-DPy was used to encapsulate C Medical technology is constantly improving to combat the harm caused by diseases to human beings. Among these techniques, the drug delivery system is critical in treating various conditions. Scientists have worked hard to develop drug carriers that are simple, accessible to process, have high stability, good biocompatibility, and are sensitive to environmental stimuli. As a proven effective drug carrier, micelles have been reported in quite a few works in the literature ,2. GenerOn the other hand, polymers with supramolecular interactions, as another way to form micelles, have attracted extensive attention in recent years . This ki60 and C70, exhibited high electron affinities and reactive exteriors and are the ideal candidate for quenching ROS [60 to fabricate fullerene-involved composites, such as through carbohydrate polymers [60 into an aqueous medium [60. However, even if covalent cross-linking provides more stability in the core, it still brings some practical limitations. On the other side, physical cross-linking via non-covalent interactions, such as H–bonding or π–π interactions, are spontaneous and can be accomplished under relatively mild conditions [60, especially through non-covalent interactions, has a significant value in expanding the application of fullerenes.Free radicals, a critical species in nature, are involved in many functions of the organism to maintain good health. Reactions involving free radicals can also be observed in other fields, such as atmospheric and polymer chemistry. Even though our bodies produce some antioxidants, excessive free radicals may still affect the biosystem. Reactive oxygen species (ROS), such as hydrogen peroxide and hydroxyl radicals, may cause severe and irreversible damage to tissues and organs . Efficiehing ROS . Howeverpolymers , silver polymers , and metpolymers . The hybpolymers ,15,16. Apolymers . Wang ans medium . This conditions . TherefoN--yl)propanamido)pyridin-2-yl)undec-10-enamide (U-DPy) was a suitable functionality to form supramolecular polymers due to its sextuple complementary hydrogen–bonding interaction. The strong H–bonding interaction can be proved by 1H NMR varian temperature experiment and the 1H NMR titration experiment, which is stronger than other base pairs such as adenine-thymine (two-point hydrogen bonding system) and diaminopyridine-uracil (three-point hydrogen bonding system) [Ka could lead to new supramolecular polymers in bulk as well as in solution [Ka) of the DAP:DAP complex was 170 M−1 [6 M−1 [Ka between the two hydrogen bonding pairs led to totally different properties of two PNIPAAm derivatives, such as the value of a lower critical solution temperature (LCST), particle sizes , kinetic stabilities under surfactants, and micelle morphologies [Tg, the supramolecular aggregation in the GPC trace, the decrease in LCST, and the increased stability against long-time SDS treatment. Owing to the strong self-complementary hydrogen–bonding interaction, PNI-U-DPy exhibits excellent micelle stability and drug loading capacities, which are suitable for application as a drug carrier. We have studied PNI-U-DPy as a carrier to load planar compounds such as doxorubicin (DOX). It was proved that the loaded micelle could successfully endocytose cancer cells [60, which is difficult to load and hard to use without chemical modification. The self-assembly of hydrogen bonding moieties and phase transition behaviors of PNI-U-DPy lead to stable micellar structures in aqueous solutions. These micelles show good C60 encapsulation capabilities and can effectively release C60 upon heating. The loaded micelles (PNI-C60) exhibit hydroxyl radicals and scavenging activities at a low dosage and could even be enhanced by ramping the temperature. Such results suggest the micelle system may have the potential to use as a vehicle for C60 radical scavenger and provide the possibility of fullerene dispersion and delivery, which expand its application on biomedical applications.Polymers with supramolecular interactions have been developed as an ideal strategy to form physically crosslinked micelles. It was proposed that  system) . It was solution . In the solution . The ass 170 M−1 , and U-D1 [6 M−1 . The sighologies . The strer cells . However60 (purity 99.5%) was purchased from Alfa Aesar . Organic solvents were purchased from TEDIA  and used as received. DMF was distilled over CaH2, and THF was refluxed with sodium lumps and distilled to remove water. All other reagents not mentioned were obtained from Sigma–Aldrich , Alfa Aesar , Showa , or TCI  without further purifications.The supramolecular polymer PNI-U-DPy was synthesized and characterized in the previous literature . C60 (pun), weight average molecular weight (Mw), and polydispersity (PDI) was investigated by Waters 510 gel permeation chromatography (GPC) system equipped with three UltrastyragelTM columns , and Dimethylformamide (DMF) was used as the eluent at a flow rate of 1.0 mL min–1 at 50 °C. Polystyrene standards with a narrow PDI were used to calibrate the GPC system. UV-Vis spectra were recorded by HP 8453 spectrometer in the concentration described in the following paragraph. The measurements proceeded with a scanning speed of 200 nm/min and UV-Vis bandwidth of 1.5 nm. The recording wavelength ranged from 190 nm to 1100 nm with a sampling interval of 0.9 nm. Each sample was measured three times to make sure the result was consistent. Dynamic light scattering (DLS) measurements were performed by Brookhaven Instrument Corporation 90-plus at a scattering angle of 90°. C60-loaded micelle samples for DLS measurement were prepared in 1,1,2,2-tetrachloroethane (TCE) and first dialyzed against N,N-Dimethylacetamide (DMAC), then dialyzed against DI water. Transmission electron microscopy (TEM) images were recorded using an FEI Tecnai T12 microscope operating at 120 kV, and the Image J program was utilized to calculate the number and diameter of micelles from TEM micrographs. Samples for the TEM observation were prepared using micelle dispersant in DI water, which was dropped onto the carbon-coated copper grids and dried under 10 °C for 72 h. The sample of the PNI-U-DPy micelle was stained with RuO4, and the C60-loaded micelle samples were observed without stain. The micelle morphologies were observed with Hitachi S-4200 field-emission scanning electron microscopy , and the samples were sputtered with Pt before imaging. Wide-angle-X-ray scattering (WAXS) patterns were performed using the wiggler beamline BL17A1 of the National Synchrotron Radiation Research Center (NSRRC), Taiwan, and the radiation with a wavelength of 1.33 Å was carried out under room temperature. Atomic force microscopy (AFM) images were recorded in tapping mode with Dimension 3100  equipped with silicon cantilevers  at room temperature. The scan rate was 0.5 Hz with a tip velocity of 5 μm/s. The resonance frequency was 394.8 kHz, and the spring constant was 42 N/m. All images were subjected to a first-order plane-fitting procedure to compensate for the sample tilt.The molecular weight information, such as the number of the average molecular weight  for 48 h, then dialyzed against DI water for another 48 h to remove the organic solvent. The resulting dispersion was centrifuged at 1000 rpm for 10 min to give a transparent supernatant. The supernatant was lyophilized for 24 h, then redispersed and diluted with DI water or a pH 7.4 Phosphate buffered saline (PBS) solution.To prepare C60 in TCE  and encapsulation efficiency (EE) of the micelles were determined using Equations (1) and (2):60-loaded micelles, we measured the UV absorption of Rhodamine-B (Rh-B) in the Fenton reaction. Based on Equation (3), hydrogen peroxide could dissociate into hydroxide ions (OH−) and hydroxyl radicals (●OH) at low pH in the presence of ferrous ions (Fe2+) [To determine the interception of hydroxyl radicals by the Cs (Fe2+) ,23.(3)F4 solution with the ratio of 2.8:0.2 (v/v). The calibration curve was set up by measuring the UV absorption at 554 nm with different concentrations. The radical scavenging experiment was carried out by mixing 0.012 mM Rh-B solution (2.8 mL), PNC60, 25 mM FeSO4 (0.1 mL), and 6.25 mM H2O2 (0.1 mL) together. The UV absorption was measured and compared to the calibration curve described above to calculate the scavenging activity. The molar concentration of PNC60 is defined by C60. The percentage inhibition was calculated with Equation (4) using the following expression [control = absorbance of the control  and Asample = absorbance of samples . Hydroxyl radical (●OH) scavenging activity under different temperatures was measured with a similar procedure. However, the mixture’s temperature was kept at a specific value  for 5 min before the H2O2 solution was added. The scavenging activity was calculated with the method mentioned above.First, we prepared the Rh-B solution with different concentrations, then mixed it with 25 mM FeSOpression :(4)Inhib3, a compound containing the azide group and U-DPy hydrogen bonding motif, to give a PNIPAAm derivative with a complementary sextuple hydrogen bonding group at the side chain [Mn is 18,073. The relatively broad PDI is caused by the multimodal distribution observed at the GPC measurement, which was attributed to the formation of aggregation by the strong hydrogen–bonding interaction and has been described before [The preparation of the supramolecular polymer PNI-U-DPy and micelles were briefly depicted in de chain . The PDId before . The pend before . During d before . It also60-loaded PNI-U-DPy micelles (PNI-C60)  at 600 nm because C60 could absorb the radiation at visible light [60 releases half of C60 at 35 °C, near the original LCST of PNI-U-DPy, suggesting its suitability and non-selectivity as a drug loading and release system.To investigate the thermal responsivity after drug encapsulation, we used the same method to monitor the release profile of CPNI-C60) b. The lyle light ,27. PNI-60 encapsulation experiment by mixing the C60 solution with PNI-U-DPy solution, then removing the organic solvent and transferring the mixture to DI water through dialysis, as mentioned above. Because C60 has an intrinsic hydrophobic nature, which may aggregate into the PNI-U-DPy micelles’ hydrophobic core, we successfully encapsulated C60 during the PNI-U-DPy self-assembly process in water. The comparison of the size of the PNI-U-DPy micelles before and after C60 loading is shown in 60 was loaded into PNI-U-DPy, the resulting micelle composite PNI-C60 exhibited a broad single distribution with a PDI of 0.177 ± 0.013, which was significantly different from the result obtained from the PNI-U-DPy micelles. Additionally, the particle size increased from 170 nm to 345 nm after loading, which suggests that C60 was successfully encapsulated into the micelles. To calculate the C60-loading efficiency, we first set up the calibration curve by dissolving C60 in TCE under different concentrations and measuring the absorbances with a UV-Vis spectrometer  and (2). The DLC is 29.12%, and EE is 5.83%, comparable with the proposed PNIPAM micelles [60 encapsulating with micelles. It is difficult to find comparison standards from other studies. Thus, the excellent DLC and EE value for micelles may lead to easier control of the loading content actually needed.PNI-U-DPy can form stable micelles due to the dimmerizable side-chain U-DPy functionality. It also exhibits suitable LCST near the physiological temperature, which is especially important for drug delivery systems. Here, we proceeded with the Ctrometer . The lyomicelles  loading 60, we prepared samples by redispersing the lyophilized PNI-C60 in the water and dropping it onto silicon wafers and carbon-coated copper grids for SEM and TEM measurements, respectively. PNI-U-DPy micelles were also used to compare in TEM observation, which was stained with RuO4. The corresponding images were collected, as shown in 60 is shown in 60 micelles show different morphologies from the PNI-U-DPy micelles  since the hydroxyl radical did not generate (2O2 was added to the cell (control sample 2), the absorption decreased seriously within 10 s, suggesting that when the hydroxyl radicals were produced and oxidized, the Rh-B molecules immediately decreased (60 is present in the mixture, the absorption at 554 nm could be higher than the control sample 2, indicating that PNI-C60 may inhibit the oxidation of hydroxyl radicals. Furthermore, we also found that with the increase in the PNI-C60 addition amount, the inhibition effect became more evident (60 was calculated, as shown in 60 was added to the assay. The addition is much lower than the previous study [60 could be used as an effective inhibitor against hydroxyl radicals.The hydroxyl radical scavenging activity of PNI-Creaction . Accordireaction . Rh-B shreaction , and thegenerate a. When tecreased e. Howeve evident b–d. The us study , signify60 concentration of 0.1 mM, and the temperature was changed from 26 to 44 °C. The result was collected and calculated in 60 undergoes the phase transition process, rapidly releasing the encapsulated C60 into the mixture and then triggering the fullerene’s inhibition mechanism. Therefore, the results mentioned above give a conclusion that PNI-C60 has the potential to act as an antioxidant with thermo-responsivity and a controlled release manner.As described in the above paragraph, PNIPAM is a thermo-responsive material, and its LCST could be adjusted by introducing U-DPy functionalities into the polymer side chain. Additionally, the C60-loaded micelles exhibit thermo-responsivity since the size of the micelles decreases after heating, which was proved by DLS and electronic microscopy observations. We continued to explore how the temperature change affected the scavenging property. The experiment was carried out at a PNI-C60 with high loading content and entrapment stability, which is seldom reported by unmodified fullerene and polymeric micelle systems. DLS, TEM, SEM, and WAXS observation results suggest that C60 has been effectively loaded into a micelle to form a complex hybrid PNI-C60. The encapsulated C60 exhibits a crystalline behavior inside the supramolecular micelles. The behavior of PNI-C60 was investigated under several variable temperature measurements and gave practical proof for the controlled release progress. Due to the existence of C60, the PNI-C60 complex exhibit hydroxyl radicals and scavenging activity at a low dosage and could even be enhanced by ramping the temperature. These findings show that supramolecular polymers could be a potential candidate as a C60 carrier without destroying its structure. This strategy provides the possibility for designing an efficient drug delivery system for those drugs that are difficult to encapsulate. It may also contribute to understanding the chemistry and biological behavior of fullerene in aqueous media, therefore offering a potential route for the fabrication of effective and eco-friendly assays for biomedical applications.In this study, it was demonstrated that the supramolecular micelles were obtained from a designed polymer PNI-U-DPy. This PNIPAM derivative contains a strong self-complementary hydrogen bonding interaction at the side chain, which constructs a physically crosslinked network and increases the stability of the micelles. The intermolecular interaction leads to an apparent change in LCST and makes it closer to the physiological temperature. It also enables PNI-U-DPy micelles to encapsulate C


Invertebrates like crabs employ their own immune systems to fight against a number of invasive infections. Anti-lipopolysaccharide factors (ALFs) are an important class of antimicrobial peptides (AMPs) exhibiting binding and neutralizing activities against lipopolysaccharides.Pp-ALF) from the blue swimmer crab Portunus pelagicus. Pp-ALF has a 369bp open-reading frame encoding a protein with 123 amino acids. The deduced protein featured an LPS-binding domain and a signal peptide. The predicted tertiary structure of Pp-ALF contains three α helices packed against four β sheets. The deduced amino acid sequence of Pp-ALF had a net positive charge of +10.75 and an isoelectric point of 9.8. Phylogenetic analysis revealed that Pp-ALF has a strong ancestral relationship with crab ALFs.This study identified and characterized a novel homolog of ALF (Pp-ALF could be revealed by in silico prediction tools. Recombinant expression of Pp-ALF was unsuccessful in the Escherichia coli Rosetta-gami expression system due to the cytotoxic effect of the peptide to the host. The toxic effect of Pp-ALF to the host was displayed by membrane permeabilization and death of the host cells by fluorescent staining with Syto9-Propidium Iodide and CTC-DAPI- FITC.Antibacterial, antiviral, antifungal, anticancer, and antibiofilm activities of  Emerging issues with antibiotic resistance in microbial pathogens have become a serious threat to the health sector . It is dLimulus polyphemus  at 37 °C at 250 rpm in an Incubator Shaker . This was added as inoculum into 250 ml LB medium with the above mentioned antibiotics and incubated at 37 °C at 250 rpm. When the culture reached OD600 of 0.5-0.7, IPTG  was added to a final concentration of 1 mM and incubated. Two ml aliquots of the cultures were taken every hour after induction, centrifuged, and the cell pellet was kept at −20 °C for the detection of the recombinant peptide by 15% SDS-PAGE examination.The mature peptide sequences of E. coli Rosetta-gami, due to the production of the peptide Pp-ALF. E. coli transformed with the pET-32a (+) vector without insert was kept as a negative control, which expresses thioredoxin (Trx). The dye suspension  was added to the cell pellets collected at 0, 1, 2, 4, and 6th-hour post-induction, incubated for 15 min in dark at room temperature and observed under Epifluorescence Microscope ( Carl Zeiss Axio observer) with Ex/Em 480/500 nm for SYTO 9 and 490/635 nm for PI.Since the turbidity of the production medium decreased post-induction and the peptide production could not be detected by SDS- PAGE, the viability of the host cells was checked by SYTO9 - PI staining. The assay was used to analyze the live (membrane intact)/dead (membrane compromised) condition of the expression host Pp-ALF) were sampled at 0, 2, and 6 and incubated with 900 μl of CTC for 90 min at 37 °C. An aliquot (10 μl) of the reaction mix was transferred to a poly L-lysine-coated glass slide. The slides were washed with sodium phosphate buffer, and 1ml of DAPI solution (10 μg/ml in PBS) was added and after 30 min at 30 °C. The DAPI solution was removed and the slides were rinsed again with sodium phosphate buffer. To the cells, FITC was added (6 mg/ml in sodium phosphate buffer) and incubated at 30 °C for 30 min and washed as mentioned above. The slides were then examined under a Fluorescence microscope .In order to observe the cells, both live/dead and membrane permeabilization caused by the peptide, if any in the expression host, triple-staining was done . FluorocP. pelagicus hemocyte by reverse transcription. Hereafter, the ALF is referred to as Pp-ALF (GenBank ID: OP009359). A similarity search using BLASTn and BLASTp algorithms confirmed the cDNA sequence coming under the ALF family of antimicrobial peptides. Upon BLASTp homology search Pp-ALF showed 95.89 % similarity with Portunus trituberculatus ALF (GenBank ID: ADU25043.1) followed by Scylla paramamosain ALF (GenBank ID: AFI43796.1) with 89.04% similarity. The putative signal peptide was identified at the N-terminal sequence, defined by SignalP software, with the cleavage site between amino acid positions 26 and 27 (CEA-QY). The signal peptide is followed by a highly cationic 97 amino acid mature peptide sequence comprising 22 amino acid long lipopolysaccharide (LPS) binding domain.A 369bp nucleotide sequence coding the complete cDNA sequence of ALF encoding 123 amino acids  of 9.8. The mature region of the sequence has aforesaid parameters as 11.29 kDa, +9.75, and 10.17, respectively. The cationic nature of the Pp-ALF was mainly contributed by Lys (7%), Arg (9%), and His (2%) against negatively charged amino acids like Glu (3%) and Asp (6%). The predicted half-life of Pp-ALF in mammalian reticulocytes is about 30h, in vitro and >10h in E. coli, in vivo. The grand average hydropathy value (GRAVY) of the peptide was estimated to be −0.29 and the Wimley white whole residue hydrophobicity was 12.87. Kyte & Doolittle plot revealed the hydrophobicity; the residues from 5 to 21 (-VVAGLCLALVVMCLYLP-) in the signal region exhibited the highest hydrophobicity  showed the highest hydrophobicity in the mature domain. The amino acid distribution resulting in amphipathicity is illustrated by the helical wheel diagram  involved in the internal disulfide bond formation were totally conserved in Pp-ALF  (95.89%) in terms of amino acid composition. Pp-ALF has Ala at the 33rd position, which is substituted by Thr in P. trituberculatus, similarly, Thr is substituted with Ile at the 54th position. A consensus pattern of WCPGWA (T) was also observed in all ALFs. The evolutionary relationships with other crustacean lineages were found by phylogenetic analysis. Bootstrapped method using deduced amino acid sequences was used to elucidate the ancestral relationships  of −123.30 kcal/mol. This indicates the structural stability of the stem-loop structure of mRNA  as template. The template shared 100% confidence with Pp-ALF. The 3D structure  and 2 (Lys 67-Trp 75) are linked by a conserved disulphide bond (Cys55-Cys 76) forming the highly cationic  and amphipathic β hairpin loop structure, the LPS binding domain. The highly cationic LPS domain permits the peptide to effectively interact with negatively charged bacterial membranes. Structure validation of Pp-ALF by Ramachandran plot  classifier. iAMPpred identified Pp-ALF as antibacterial, antiviral, and antifungal with 0.96, 0.96, and 0.98 SVM score, respectively. SVM score near to 1 is identified as a peptide with high therapeutic potential. Even though Pp-ALF was predicted to exhibit antimicrobial potential, the region with high aggregation propensity towards microbial membrane was predicted by AGGRESCAN. There are two hotspots predicted for Pp-ALF; the first hotspot (HS) area with a normalized value of 0.745 was found inside the signal peptide domain (GVVAGLCLALVVMCLYL), and the second HS was inside the mature region (DALVASILGKLTGL) with a normalized value of 0.347.CAMP server predicted Pp-ALF was predicted with anti-angiogenic activity by “AntiAngioPred” with an SVM score of 0.82 indicating its anticancer property. Also, the average antigenic propensity of Pp-ALF was found as 1.0366 by the “Immunomedicine group.” The server predicted 5 antigenic sequence regions in Pp-ALF, i.e., 4 to 38 residues (GVVAGLCLALVVMCLYLPQPCEAQYDALVASILGK), 49 to 57 (DFMGHTCYF), 64 to 70 (RRFKLYH), 72 to78 (GKFWCPG), and 112 to119 (ITQQDASL). So, Pp-ALF could be used as an immunogen with prediction values of 0.316 (AntiTbpred) and 0.574 (AIPpred) as anti-tubercular and anti-inflammatory peptide. Biofilms provide survival sites for opportunistic pathogens. The LPS domain of Pp-ALF was predicted as an antibiofilm agent (SVM score −0.83). The ability to distinguish between an antigenic cell and host cell is an important property of an ideal AMP. HemoPred predicted the LPS domain of Pp-AF as non-hemolytic. This property leads to the safe application of the peptide in the biological system.The LPS domain of Pp-ALF was predicted to be CPP (cell-penetrating peptide) with a positive SVM score of 0.01. CPPs are made up of basic amino acids like arginine and lysine, and they can cross membranes to obtain access to the cell’s core. Pp-ALF consists of a total of 17 arginine and lysine residues in the mature region of the peptide. This was confirmed by “DNA binder”, which gave the Pp-ALF a positive score of 1.25, indicating it as a DNA-binding protein. Pp-ALF possesses immunomodulatory activity as predicted by “VaxinPAD” with an SVM value of 0.97. So, Pp-ALF has the potential to stimulate the innate immune system as an adjuvant. In immunotherapy, pro-inflammatory peptides (PIP) have been employed as anticancer agents, antibacterial agents, and vaccines. Pp-ALF has been predicted as pro-inflammatory peptide (PIP) with a probability score of 0.7633. The projected value of the codon adaptation index, which represents the likely success of heterologous gene expression was, 0.57 in Escherichia coli, 0.58 in Saccharomyces cerevisiae, and 0.6 in Pichia pastoris. The peptide rank score was calculated to be 0.99, indicating the high bioactive potential of Pp-ALF.Peptides are divided into two types based on their amino acid composition; cell-penetrating and non-cell-penetrating peptides. E. coli (pET-32a- Pp-ALF) was found decreasing post-induction with IPTG. The cell lysate of the expression host E. coli when analyzed by SDS-PAGE did not display the presence of the recombinant peptide Pp-ALF (29.08kDa with Trx tag)   Fig. . However2a) Fig. .Fig. 10TPp-ALF. The recombinantly expressed Pp-ALF causes the death of the expression host, and this was observed on staining. The cells were green fluorescent at the 0th hour since they were metabolically active. From the 1st hour post-induction itself, the cytotoxic effect on host cells was clearly visible and from the 2nd hour onwards complete cell death was confirmed by red fluorescence due to the internalization of propidium iodide stain  staining of the host cells revealed the death of the cells during the production of in Figs.  and 13. th h) at the time of IPTG induction as evidenced and confirmed by CTC-DAPI–FITC staining. Reduction in viable cells and membrane permeabilization was observed at the 2nd h. Viable cells were not observed at the 6th h, and only membrane-permeabilized/dead cells were detected. This concludes that the peptide Pp-ALF causes death to the host cells at the initial stages of production itself. The peptide also causes a high degree of cellular clumping which was also observed in cells at the 6th h , ALFPm3, (Penaeus mondon) SpALF4 (Scylla paramamosain), and SpALF7 [Pp-ALF has a stronger association with crab ALFs than shrimp and crayfish ALFs.Due to the emergence of antibiotic-resistant microbes, the significance of alternate compounds like AMPs to combat infections has been increased . In the hobicity –26. Pp-Ag domain , 27. Thed SpALF7 –31. The d SpALF7 . On phylPp-ALF possesses 8 hydrophobic amino acids including one tryptophan. It has been demonstrated that the ALFs with high pI value, bind with LPS inhibiting the proliferation of Gram-negative bacteria [Pp-ALF, nine positively charged amino acid residues are present with a high pI of 10.54. Rosa et al. [S. paramamosain, SpALF1 and SpALF2 with high pI showed antimicrobial activity against both Gram-negative and Gram-positive bacteria [The LPS-binding domain of bacteria –34. Insia et al.  reportedbacteria .Pp-ALF had a net charge of +10.17 for the mature peptide indicating the high cationicity of the peptide. It is generally accepted that the initial interaction of the AMP with the negatively charged membrane surface of the bacterium is predominantly caused by cationicity [Pp-ALF embedded with five arginine and three lysine residues contributing 23 and 14% of the total amino acids, respectively, in the LPS region indicating its potential role as an antimicrobial agent. FcALF2 (Fenneropenaeus chinensis ALF) and PmALF3 (Penaeus monodon ALF) with five and six positively charged amino acids in the LPS domain have shown strong antibacterial activity [Pp-ALF shares important characteristics of LALFs (Limulus ALF), with the hydrophobic N-terminal sequences and the concentration of positive charges in the disulphide loop that are crucial for their antimicrobial effect [The biological functions of ALFs mainly depend on the LPS domain that interacts with the negatively charged LPS . Positivionicity , 43. LPSactivity , 44. Pp-l effect , 46.Pp-ALF was found to possess 41% hydrophobicity as per APD analysis. Hydrophobicity controls the degree to which a peptide can partition into the lipid bilayers and therefore play an important role in its activity [Scylla paramamosain was higher (51.4%) than that of rSpALF7 (41.5%), which might be one of the reasons why sSpALF7 showed a broader antibacterial spectrum. Analysis of the Pp-ALF sequence using the Kyte-Doolittle plot exhibited a significant presence of hydrophobic amino acids concentrated in the first 17 residues (-VVAGLCLALVVMCLYLP-) of the mature peptide. This demonstrates that these amino acids are components of the alpha helix and may have the ability to traverse a lipid bilayer of the microbial membrane [Pp-ALF was predicted to possess antibacterial, antifungal, and antiviral activities, and it was found to have high therapeutic potential according to the prediction score. Six recombinant PtALF proteins could prevent the growth of specific Gram-positive, Gram-negative bacteria, or fungi in the swimming crab P. trituberculatus [activity –49. Hydractivity , 50. Thes higher .4% than rculatus , 52.P. monodon showed inhibition against herpes simplex virus type 1, human adenovirus respiratory strain, and WSSV replication [Fenneropenaeus chinensis could inhibit WSSV replication [P. trituberculatus, showed minimal effective concentrations (MECs) of 2.11 μg/mL and 1.95 μg/mL against the yeast Candida albicans, respectively, demonstrating promising antiviral and antifungal activity against yeasts and viruses [P. monodon [Fenneropenaeus chinensis) resulting in the leakage of cytoplasm [Marsupenaeus japonicas also exhibited antibacterial activity [Pp-ALF is predicted as a cell-penetrating peptide and therefore might cause pore formation resulting in the leakage of cell contents.ALF from lication , 53. LBDlication . Crab-AL viruses . ALFPm3  monodon  showed aytoplasm . MjALF-Dactivity . Pp-ALF Pp-ALF is also predicted to have antiangiogenic properties by the Anticp and antiangiopred server with a significant prediction score.Synthetic SALF (shrimp ALF) from black tiger shrimp displayed anticancer properties in HeLa cells when tested in mice. SALF disrupted the tumor cell membrane triggering apoptosis . Pp-ALF E. coli BL21. The fusion approach enhances solubility and prevents toxicity and cell disintegration in the expression host. The recombinant expression has greater benefits than high-cost chemical synthesis and low-yield natural extraction. Due to the potent antibacterial properties displayed by Pp-ALF in silico, recombinant production was attempted using the E. coli expression system. However, the peptide was found to be toxic to the host cells since post induction with IPTG, the cells started dyeing which might be due to the production of the peptide Pp-ALF. rMnALF4 (recombinant ALF from Macrobrachium nipponense) also showed toxicity in E. coli expression system; so it was produced in P. pastoris with a high yield. Additionally, Salmonella typhi, Shigella sp., Staphylococcus aureus, Pseudmonas fluorescens, and Salmonella gullinarum, all of which are extremely pathogenic for humans, were sensitive to rMnALF4 [Pp-ALF production was toxic to the host, which was proved by live dead SYTO 9-PI staining and triple staining. Death of the expression host was observed from the first hour onwards after IPTG induction.The majority of the studies have concentrated on the production of AMPs in bacteria due to the availability of well-defined plasmid vectors and the practical significance of large-scale peptide production through fermentation. However, since some of these peptides are toxic to the expression host, direct expression of AMPs is typically challenging . As a re rMnALF4 . Pp-ALF P. pastoris is widely used in the heterologous expression of antimicrobial peptides, because of its potent secretion and glycosylation property. Anti-lipopolysaccharide factors from P. mondon [Macrobrachium rosenbergii [Macrobrachium nipponense [Litopenaeus vannamei [P. pastoris. ALF from M. nipponense could not be expressed in E. coli [Pp-ALF could not be expressed in E. coli. The reason for this differential antibacterial effect displayed by E. coli could not be explained.The eukaryotic yeast expression host, . mondon , Macrobrenbergii , Macrobrpponense , and Litvannamei  were suc E. coli , and in E. coli system, is not typically used [P. pastoris expression system when a signal peptide is linked to the foreign protein at its N-terminus [Pp-ALF was toxic to E. coli cells, an alternate expression system like P. pastoris would be a viable option. The success of heterologous expression predicted by the codon adaptation index showed that P. pastoris system has the highest chance for expression of Pp-ALF. Synthetic biology is also another option for the synthesis of these molecules [P. pelagicus with potent antimicrobial activity predicted in silico and displayed by the death of the host E. coli DH5 alpha cells on heterologous expression.Recombinant proteins can take on specific spatial structures and post-translational modifications when they are expressed by the eukaryotic expression system. These changes would better replicate the recombinant proteins’ natural state in living organisms, and the structure and modification are crucial for protein function . For thelly used , 61. Altlly used . Conditilly used . Recombiterminus . Since Polecules . The useP. pelagicus, Pp-ALF revealed that it could be a potent molecule with respect to antimicrobial, anti-inflammatory, and anticancer activity. The prokaryotic expression system E. coli was not found suitable for the recombinant expression of Pp-ALF due to its cytotoxic effect on the host cells. Eukaryotic expression systems would be a preferred option for the recombinant production of Pp-ALF.Functional prediction of the anti-lipopolysaccharide factor from


Surveillance data shows a geographical overlap between the early coronavirus disease 2019 (COVID-19) pandemic and the past Q fever epidemic (2007–2010) in the Netherlands. We investigated the relationship between past Q fever and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in 2020/2021, using a retrospective matched cohort study.In January 2021, former Q fever patients received a questionnaire on demographics, SARS-CoV-2 test results and related hospital/intensive care unit (ICU) admissions. SARS-CoV-2 incidence with 95% confidence intervals (CI) in former Q fever patients and standardised incidence ratios (SIR) to compare to the age-standardised SARS-CoV-2 incidence in the general regional population were calculated.Among 890 former Q fever patients (response rate: 68%), 66 had a PCR-confirmed SARS-CoV-2 infection. Of these, nine (14%) were hospitalised and two (3%) were admitted to ICU. From February to June 2020 the SARS-CoV-2 incidence was 1573/100 000 (95% CI 749–2397) in former Q fever patients and 695/100 000 in the general population . The incidence was not significantly higher from September 2020 to February 2021.vs. the general population during the period with widespread SARS-CoV-2 testing availability (September 2020–February 2021). This indicates that former Q fever patients do not have a higher risk of SARS-CoV-2 infection.We found no sufficient evidence for a difference in SARS-CoV-2 incidence or an increased severity in former Q fever patients  In February 2020, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus causing coronavirus disease 2019 (COVID-19), reached the Netherlands and started spreading. SARS-CoV-2 is a respiratory virus, with symptoms varying from mild respiratory symptoms to severe pneumonia and death [The COVID-19 epidemic started in several regions in the Eastern part of the province Noord-Brabant in the Netherlands. These were the same regions where a large Q fever epidemic occurred from 2007 to 2010. A preliminary study on a small group of COVID-19-related hospital admissions (mostly inhabitants of Noord-Brabant) showed a slightly higher seroprevalence of Q fever (16%) than previously estimated for high-risk areas (12–15%) .Coxiella burnetii, which can cause symptoms ranging from mild respiratory complaints to severe pneumonia and death. However, most infected people remain asymptomatic (60%) [Q fever is a disease caused by the bacterium ic 60%) . An acutic (60%) , 6 or lo0% . An aic (60%) .The Q fever epidemic from 2007 to 2010 was most severe in the South-East region of the Netherlands, with a peak in cases each spring. In total, over 4000 acute Q fever cases were reported in this period. This is an underestimation of the true incidence, as not all Q fever infections were detected or reported .As surveillance data show a geographical overlap between the regions of the Q fever epidemic in 2007–2010 and the first wave of the COVID-19 epidemic in 2020 [A retrospective matched cohort study was performed using newly collected data on COVID-19 among patients who were diagnosed with acute Q fever in 2007–2009. Additionally, we used mandatory notification data, collected from the notification data base called OSIRIS, of SARS-CoV-2 patients reported from February 2020 to February 2021 who lived in the same region. During the first period of the COVID-19 epidemic in the Netherlands, a strict policy governed who was tested, as test capacity was limited. Not everyone with symptoms was able to get tested. From the first of June 2020, however, all inhabitants of the Netherlands could get a PCR test  if they experienced symptoms that could be explained by SARS-CoV-2. To account for a potential difference in data gathered before and after the implementation of unrestricted testing, we selected two time periods. Period one was 1 February 2020–1 June 2020, and period two was 1 September 2020–1 February 2021. We omitted July and August, as the SARS-CoV-2 incidence was very low in that period.n = 1447). Q-HORT was a four-year follow-up study aiming to detect chronic Q fever cases amongst patients diagnosed with acute Q fever between 2007 and 2009. The case definition for acute Q fever is described in the study of Wielders et al. [The study population consisted of former Q fever patients who participated in the Q-HORT study between 2011 and 2013  and who s et al. . The queTo compare the former Q fever patients to the general population, data for all persons testing positive for SARS-CoV-2 from February 2020 to February 2021 were extracted from OSIRIS. They included variables on age, gender, region, hospital and/or ICU admission and death.For comparing the SARS-CoV-2 incidence in former Q fever patients we selected Region A, which consists of 12 municipalities in the South of the Netherlands (Noord-Brabant) where most Q-HORT participants lived . Age-speC. burnetii infections in the general population in Region A. Region B consists of 14 municipalities in the Mid-East of the Netherlands outside the area where the Q fever epidemic happened, but with a similar SARS-CoV-2 incidence from February to June 2020 as Region A (For the severity comparison we selected a second region (Region B), since COVID-19 severity might be underestimated due to more asymptomatic Region A . To compDescriptive statistics were used to summarise patient age, gender and underlying comorbidities. SARS-CoV-2 incidences per 100 000 population were calculated, including 95% confidence intervals (CI) for incidence in the former Q fever patients. The incidence in Region A was age standardised according to the age distribution in the former Q fever patients. The SARS-CoV-2 incidence in former Q fever patients who responded by filling in the questionnaire was compared to the age-standardised SARS-CoV-2 incidence in the general population in Region A for the total study period (February 2020–February 2021), period one (February–June 2020) and period two (September 2020–February 2021). Participants of the Q-HORT study who died with SARS-CoV-2 were not included in the calculations of the SARS-CoV-2 incidence in former Q fever patients, because the data were collected differently and might have biased the calculated incidence. Furthermore, we used Poisson regression (R-package popEpi) to calculate the standardised incidence ratio (SIR) with 95% CI for SARS-CoV-2 in the former Q fever patients compared with the general population in Region A.To determine the severity of COVID-19, conditional logistic regression  was conducted to compare the risk of hospitalisation, ICU admission and death between SARS-CoV-2 infections in former Q fever patients (including those who died with SARS-CoV-2) and matched SARS-CoV-2 cases from the general Dutch population living in Region A or B. For each outcome of interest, odds ratios (OR) with 95% CI were calculated. Where possible, we calculated OR and 95% CI for period one and two and Region A and B separately. Data were analysed using R 4.0.2 Statistical Software.Former Q-HORT participants  who consOf the 1447 former Q-HORT participants, an updated Dutch address could be retrieved for 1304, of whom 890 (68%) completed the questionnaire that they received on 11 January 2021. In addition, we identified 17 Q-HORT participants who died in 2020–2021. Of these, ten were reported to the GGD Hart voor Brabant because of a laboratory-confirmed SARS-CoV-2 infection. They were therefore added to our subjects and included in the severity analysis.Of the 900 total, 497 (55%) were male, and the median age was 63 years (54–70 interquartile range (IQR)). The majority had one or more underlying comorbidities (74.3%). The most frequently reported chronic diseases were cardiovascular disease , Q fever fatigue syndrome , chronic Q fever , chronic lung condition  and immunodeficiency . A small number of respondents were pregnant in 2020–2021 . Excluding Q fever fatigue syndrome and chronic Q fever, 63.3% of 900 former Q fever patients had one or more underlying comorbidities. Of the 900 subjects, 201 (22.3%) reported symptoms possibly related to COVID-19, but had no laboratory confirmation.n = 890) being 7415/100 000 (95% CI 5627–9205) compared to the age-standardised incidence of 5786/100 000 in the general population   .Table 1Of 900 former Q fever patients, 76 had a laboratory-confirmed SARS-CoV-2 infection (8.4%). Of these, 16 (21.1%) were hospitalised, five (6.6%) admitted to the ICU and ten (13.2%) died.n = 21; 28.8%) compared to the second period . The former Q fever patients showed a higher percentage of hospital admissions, ICU admissions and death  compared to matched SARS-CoV-2 cases from the general population in Region A  and in Region B  ; the median age was 63 (IQR 54–71), and 52 reported one or more underlying comorbidities (71.2%). A minority of cases received a positive test result in the first period (ctively) . HoweverThe study shows a statistically significant increase in the incidence of SARS-CoV-2 in former Q fever patients compared to the general population in Region A for period one (restricted testing policy). However, no statistically significant rise in incidence was seen for period two  or for the entire study period from February 2020 to February 2021. In no period was there sufficient evidence for a more severe course of COVID-19 in former Q fever patients.There are several potential explanations for the higher incidence of SARS-CoV-2 in former Q fever patients in period one. First, it could be related to the effects of acute Q fever in the past. In that case, however, we would have expected a higher incidence in the second period as well, which was not observed. Second, more testing might have occurred in former Q fever patients, especially during the first period, if their disease course was more severe compared to the general Dutch population. The matched analysis on severity outcomes , however, showed no such differences in either region or time period.C. burnetii infection, or whether they were already present before the Q fever diagnosis, and/or whether they made these individuals more susceptible to get Q fever.Finally, up to 1 June 2020 there was a restricted policy on who could get tested. More former Q fever patients had underlying illnesses  than individuals in the general Dutch population (57% on 1 January 2019) . Since fIt is important to bear in mind that there is a potential bias for a higher incidence and more rapid spread of SARS-CoV-2 in Region A, where the former Q fever patients live. It has poorer air quality than other provinces in the Netherlands , which mTo our knowledge, Q-HORT is the largest Q fever patient cohort worldwide that was available for follow-up. This, in combination with the high response rate to our questionnaires (68%) gives us a good overview of the COVID-19 incidence and severity in this group of patients.However, the study has several limitations. First, the Q-HORT study group previously participated in a scientific study and consented to participate in further research, suggesting that they are a select group. Findings based on such a group may have limited application to former Q fever patients in general. Moreover, their willingness to participate could reflect a Q fever episode that was particularly severe and/or resulted in more long-term problems than usual. This applies even more to the former Q fever patients who participated in this study since they consented and participated in scientific research for a second time. Second, persons who experienced severe Q fever and/or who have long-term related problems might be more cautious and more compliant with COVID-19 measures set by the government. This could result in fewer cases of COVID-19 in former Q fever patients. Third, recall bias may have occurred, with former Q fever patients over-reporting illness episodes because they are unusually focused on health problems and symptoms. Alternatively, they might under-report episodes, having forgotten their illness. The latter is unlikely, as COVID-19 is a fairly new disease and we expect people to remember a positive test result, which we used as outcome in this study. Fourth, we cannot exclude the possibility that a former Q fever patient with a positive SARS-CoV-2 test was selected as its own match from the notification database. We were not able to identify the notified cases and compare them to the former Q fever patients due to privacy restrictions. It is highly unlikely that a former Q fever patient is its own match, as there were many SARS-CoV-2 cases, but we cannot be certain of this.Finally, due to the low number of hospital admissions, ICU admissions and deaths, results for severity per region and time period must be interpreted with caution. Especially for period two, the conditional logistic regression could not be performed for certain subgroups due to low numbers.In general, results should be interpreted with caution given the few cases with SARS-CoV-2 in our study population. Also, other explanations exist for the higher SARS-CoV-2 incidence in period one, and further research will investigate them. The possible relationship between COVID-19 severity, air quality and livestock farms is under investigation by the RIVM [